PROJECT SUMMARY/ABSTRACT Accurate and timely detection of circulating autoantibodies against pancreatic islet antigens is critical to both research and clinical care for patients with type 1 diabetes (T1D). However this measurement remains highly variable across commercially available assays, and such assays also may not adequately detect particularly low but clinically relevant levels of circulating autoantibodies. This deficit translates into missed opportunities for both the timely initiation of the most appropriate treatment regimens and to support much needed research into novel and improved disease-modifying interventions. Additionally, large-scale public health screening efforts for T1D are hampered by the low throughput nature of current bioassays, and/or their requirement for expensive and specialized instrumentation. We have developed a proprietary patent-pending PCR-based technology termed Antibody Detection by Agglutination-PCR (ADAP). ADAP is a high-throughput assay that can be used for simultaneous detection of multiple antibodies while requiring only a very small amount of patient serum (2 ?L). ADAP also detects autoantibodies with 1,000 to 10,000 times greater sensitivity than the currently used immunometric and radio-immuno assays, and can be readily integrated into common quantitative PCR (qPCR) workflows using pre-existing instrumentation that is available at many hospitals, clinics and public health screening sites. Importantly, ADAP represents a significant departure from less effective PCR-driven platforms such as immuno-PCR, overcoming many of the deficits inherent to this class of assays to afford a high-throughput, ultrasensitive, robust, reliable, and specific detection method. In this Phase 1 application, we propose to develop an ADAP-based assay kit for detection of the four autoantibodies that form the basis for T1D diagnosis. The corresponding GAD65, IA-2, insulin, and ZnT8 antigens will be barcoded by conjugation to unique single-stranded DNA sequences. Agglutination of an antigen upon incubation with its cognate antibody brings the DNA sequences near to each other. An appropriate ?bridge oligo? is then supplied which, upon ligation, affords an amplifiable DNA duplex. This antigen-autoantibody binding event provides a PCR amplicon that enables ultrasensitive detection with minimal background signal. Next we will use these appropriately validated reagents for analysis of patient serum samples in the context of T1D, both before and after diagnosis. Finally, it is increasingly appreciated that high-affinity autoantibodies are privileged indicators of disease severity. In anticipation of the diagnostic value of this new finding, we also propose to create an innovative variant of the ADAP assay which we term ?ADAP to enable high throughput quantification of high-affinity autoantibodies. Deployment of such an assay may allow more accurate determination of T1D prognosis, and enable improved choices of therapeutic interventions. In summary, we seek to establish the ADAP T1D assay kit not only as an effective alternative to current T1D diagnostic platforms, but one with significantly expanded capabilities in terms of sensitivity, speed, reproducibility and multiplexability that can be cost-effectively integrated into existing laboratory and clinical workflows.